The invention relates to a cooling chamber cooled by liquid nitrogen (LN.sub.2) and swept with gaseous nitrogen (GN.sub.2) and intended for producing thin, particularly ultra-thin, sections for microscopic and electron microscopic examination.
According to the current state of the art, LN.sub.2 cooled and GN.sub.2 swept or GN.sub.2 filled cooling chambers are preferably used in the field of cryo-ultramicrotomy for electron microscope examination of viscoplastic anti-adhesive or biomedical objects, instead of the previously used cooling cartridges or cryostat systems (cf. H. SITTE, Instrumentation for Cryosectioning, in: "Electron Microscopy 1982, Vol. 1, 10th Int. Congress on Electron Microscopy, Hamburg, pp. 9 to 18; H. SITTE and K. NEUMANN, Ultramicrotomes and appliances for ultramicrotomy, Geratetechnik--Funktion--Zubehor, in: G. SCHIMMEL and W. VOGELL: A collection of methods for electron microscopy, contribution 1.1.2, Wiss. Verl. GmbH, Stuttgart 11, 1984, 1 to 248, particularly pp. 69 to 91; C. REICHERT AG, A-1170Vienna. Catalogue 1.K.-FC 4 D-D 4/84, FC 4 D Low Temperature Cutting System according to SITTE, 1984; C. REICHERT AG, A-1170 Vienna, Catalogue 1.K.-CRYO PREP. E-8/86, Cry preparation for superior EM investigation, 1986). With the currently preferred FC 4 D system of REICHERT AG in A-1170 Vienna, a cutting space in which a frozen object and a knife are located is an open topped sheet aluminum box which is directly cooled by LN.sub.2. For this purpose, having regard to the available space with an ultramicrotome, there are on the right and left two intercommunicating sheet aluminum tanks ("a twin tank system") to accommodate about 1 liter of LN.sub.2 and these are connected to the cutting chamber. This arrangement (twin tanks with cutting chamber) makes it possible to achieve minimal temperatures which come very close to the boiling point of LN.sub.2 (-196.degree. C.), and also make it possible to work with an open cutting room ("open top work") without the risk of ice deposits in the object knife area. This situation owes much to a strong GN.sub.2 current which is constantly being vaporized from the LN.sub.2 which is used as the cryogenic medium and which is partially deflected by baffle plates to the bottom of the cutting chamber. In this way, the cutting chamber is swept from the bottom upwards by a stream of cold, dry GN.sub.2 which on the one hand cools the object and the knife and also supports therefor, while on the other it prevents the ingress of humid room air into the cutting chamber. Since with regard to the heat and cold sensitivity of the ultramicrotome mechanism, it is also necessary to avoid frost deposits as well as cold surfaces on the outside of the tank system, the system described is enclosed by an insulating layer of foamed synthetic plastic (e.g. foamed polystyrene or polyurethane) and finally a chamber wall which is thermostatically heated to room temperature and which consists of a readily heat conductive material (e.g. cast aluminum) (see inter alia H. SITTE, K. NEUMANN, H. KLEBER and H. HASSIG, Cooling Chamber for Holding Objects Requiring Work, Particularly Biological Objects, German Patent Application No. 29 06 153 C 2 of Feb. 17, 1979). Such a chamber wall not only prevents frost forming, which cannot be avoided after prolonged use even with a relatively thick layer of insulation, but at the same time it warms the GN.sub.2 flowing over the outside walls up to temperature levels which no longer have a disturbing influence on the sensitive ultramicrotome mechanism. The chamber wall is, via a resilient interlining, fixed on an assembly element (e.g. a dovetail joint), by means of which it can be mounted on the knife support of the microtome or ultramicrotome. The assembly element carries a baseplate for the cutter holder in a rigid metallic connection. In contrast, the object is now generally fixed on a bridge-like carrier element which is mounted on a preparation carrying arm of the microtome or ultramicrotome and plunges down into the cutting chamber from above (CHRISTENSEN'S "bridge", see A. K. CHRISTENSEN, J. Cell Biol. 51, 1971: 772-804). Where such cooling chambers are concerned, vital importance is attached to the way in which the tank system is filled or topped-up with LN.sub.2. As a rule, the LN.sub.2 tank is filled through a pipe from a storage tank (e.g. 50 liter Dewar tank). Since a continuous LN.sub.2 top-up is very difficult to achieve by reason of substantially varying working conditions, topping-up is intermittent and is controlled by measuring probes (e.g. thermosensitive diodes) so that topping-up with LN.sub.2 starts when there is a preselected minimal level of LN.sub.2 in the twin tanks and is stopped again when a likewise preselected maximum level is reached. After topping-up with LN.sub.2 is stopped, there is a pause in which GN.sub.2 is decocted from the LN.sub.2 present in the tank system of the cooling chamber until such time as the minimal LN.sub.2 level is again reached and another topping-up process commences. In the time elapsing between two topping-up processes, a hose connection between the Dewar storage tank and the cooling chamber becomes sufficiently heated so that for a fresh top-up with LN.sub.2 it is necessary firstly to cool down again to -196.degree. C. This causes a violent boiling process during which large quantities of GN.sub.2 are given off which in turn, as they pass through the cutting chamber, have a disturbing influence on the cutting process. Therefore, emergence through the cutting chamber of this GN.sub.2 which is initially formed during the topping-up process is prevented by an LN.sub.2 liquid barrier of the same type as a siphon normally used in a sanitary installation as a seal vis-a-vis a sewage system ("a phase separator" for separating the GN.sub.2 from the LN.sub.2 during topping up). Cooling chambers currently equipped with such "separators" react to the initial surge of GN.sub.2 during topping-up but only in a very weakened manner.
Very soon it became obvious that the minimum attainable object and cutter temperature does not in most cases represent the optimum temperature for the taking of sections. For example, in order to take sections in thicknesses of between 0.5 and 1 .mu.m from biomedical specimens which, prior to being frozen, have been treated with aldehyde and sugar solutions by the TOKUYASE method currently used for histochemistry (see G. GRIFFITH and coll., J. Ultrastructure Res. 89, 1984: 65 to 78), a temperature around -80.degree. is optimum, since at lower temperatures, the frozen material becomes too brittle and crumbly for taking sections of this thickness. If one would like to take from the same specimen, for electron microscopic investigation, ultra-thin sections in thicknesses .ltoreq.0.1 .mu.m, then this is possible only by reducing the temperature to about -100.degree. C. If it is desired to cut biomedical specimens which become amorphously vitrified by rapid cryofixation, in this amorphous state, then these section preparations must be carried out in a temperature range below -140.degree. C., since amorphously vitrified water changes to cubic-crystalline water at the devitrification temperature of -135.degree. C., so that the sensitive fine structures are destroyed. Finally, many visco-plastic and/or antiadhesive polymers (e.g. natural or synthetic rubber, TEFLON, polyethylenes) can only be cut at below their glass point, since it is only then that they attain the hard consistency needed for cutting. Many substances (e.g. silicone rubber, TEFLON) only attain this consistency in the immediate vicinity of the boiling point of LN.sub.2, i.e. around -190.degree. C.
Chambers of the described construction indeed reach ultra low temperatures without any problems, close to -190.degree. C. and are therefore outstandingly suitable for cutting amorphously vitrified biomedical objects and for cutting polymers with an extremely low glass point. However, they are only conditionally suitable for cutting biomedical specimens after a sugar impregnation according to TOKUYASU's method in the temperature range .gtoreq.-100.degree. C. It has been demonstrated that indeed the metallic object and knife support can be very rapidly heated to values .gtoreq.-100.degree. C. by means of a heating cartridge but that by reason of the violent scavenging of the object and the knife by cold GN.sub.2, the tip of the object and the edge of the knife nevertheless stay at temperatures which are far closer to the temperature of the cold scavenging gas (about -170.degree. C. at the level of the knife edge) than to the temperatures of the heated metal supports. Therefore, with such systems, one is compelled likewise substantially to heat up the GN.sub.2 scavenging gas. According to the current state of the art, this is carried out by a heated metal plate mounted in the bottom of the chamber and over which the cold stream of GN.sub.2 is directed. Thus, it indeed is possible to achieve the desired heating of the scavenging gas from -170.degree. to about -100.degree. C. and, as the process continues, usable working conditions in the temperature range of up to about -80.degree. C., but one does firstly fall foul of the limit of the potential of such systems while secondly the facility achievement is paid for by a considerable increase in LN.sub.2 consumption. The intimated limit of possibility is a kind of "vicious circle", i.e. the heating elements in the chamber, particularly the heating plate mounted in the bottom of the chamber for heating the GN.sub.2 scavenging gas, causes a more violent boiling off of GN.sub.2 from the LN.sub.2 tank, thus increasing cooling by the GN.sub.2 scavenging gas and finally causing a more pronounced output of heat in the chamber. This working cycle limits the counter-heating to GN.sub.2 temperatures of around -100.degree. C. The LN.sub.2 consumption of the system thereby increases from about 3.5 liters LN.sub.2 /hr for a cutting chamber of about 1 liter capacity without counter heating (GN.sub.2 temperature -170.degree. C.) to about 7 liters LN.sub.3 /hr with counter heating which produces a GN.sub.2 temperature of around -100.degree. C. in the same system. Since this increased LN.sub.2 consumption is endogenously produced, i.e. by the heat output from the gas heating plate in the cutting chamber, it cannot be outwardly diminished by increasing the foam insulation around the tank system. Apart from this disturbing phenomenon which in addition renders impossible section preparations in the temperature range of between 0.degree. and -50.degree. C. which is normally used for conventional cryostat systems of light-microscopic histochemistry and micromorphology in such cooling chambers, the section preparation is affected by the initial release of GN.sub.2 in the connecting tube between the Dewar storage tank and the chamber tank, despite the interposed separator systems, because these cannot prevent the wave motion of the LN.sub.2 in the twin tank which is triggered by the shock waves. In consequence, the LN.sub.2 in the twin tank briefly wets areas of the chamber walls of which the temperatures are somewhat above the boiling temperature. It necessarily follows that there is also an onset of boiling of the LN.sub.2. The GN.sub.2 which is liberated is deflected by baffle plates into the cutting chamber where it causes the temperature to drop by about 1.degree. to 3.degree. C. and as the process continues some of the microsections in the section strip may be unsuccessful. Finally, practice has shown that the assembly of heavy and all in all large-volume and extensive chambers (volume totalling over 4 liters, dimensions: height.times.width.times.depth about 15.times.25.times.15 cc) on a precision cross slide for the supporting of a microtome or ultramicrotome is not sufficiently stable. Rather, despite all manner of protective measures, in the virtually inevitable event during preparation of the sections that, a hand is placed on the chamber wall, there will be a tendency to allow a troublesome change in the relative position between the preparation and the knife which makes it virtually impossible immediately and without risk to continue a series of sections following such an interruption.